As a major effort, the newly developed difference hydrogen exchange method will be used to study structure change in hemoglobin. Individual structure changes that register in the hydrogen exchange data will be surveyed. (Already 65% of the exchanging peptide hydrogens of hemoglobin have been surveyed and 5 separate changes found.) The free energy associated with each change will be measured. Efforts will be made to identify each change with its segment of origin by reference to the x-ray structure. These efforts will utilize specific site modifications (e.g. at the reactive SH), reconstituted partially liganded (cyanomet) subunit hybrids, specific subunit C-terminal proteolysis, etc. The sequence in which these changes develop will be determined by observing the degree of development of each change as a function of increasing degree of liganding. In these latter experiments, bookkeeping of the free energy invested in each change at each intermediate degree of liganding should show how each change contributes to each of the others. Overall, this information should help to show how energy is carried through the protein in the form of structure changes, and how this structural energy is transduced into change in ligand binding energy. A collaborative project, with Prof. Annemarie Weber of this department, using our hydrogen-tritium exchange techniques, will be undertaken to study structure changes in actin and in myosin associated with the conversion of chemical to mechanical energy. The effort to identify, and measure the free energy of, different structure changes that accompany oxidation-reduction in cytochrome c may also be initiated.